There are many clinical settings where tests on the viable white blood cells or subpopulations of blood cells are desired. For instance, in the HIV/AIDS field it would be of benefit to know if specific T cell subpopulations are present in the blood and are capable of eliciting an immune response to the virus. In the vaccination clinical trial arena, it is desirable to know if an immunization induces the formation of disease fighting cells. In the field of autoimmunity it is important to both isolate pathogenic T cells and perform functional studies on these cells.
High throughput screening of white blood cells (WBCs) is desirable, particularly for investigations in personalized medicine and a patient's response to drugs. This is difficult if the starting method is gradient separation of whole blood, because this method is not easily automated. Furthermore it is desirable clinically to separate blood in a fashion that yields different populations of blood cells each time with different starting viabilities. There is an unmet need for automation of whole blood separation of WBCs with reproducibly high viability, yield, and purity.
The functional and cell surface traits of white blood cells cannot be easily examined without these cells being physically separated from the red blood cells (RBCs). However, functional and quantitative examination of WBCs, or subpopulations of WBCs in blood samples has been hampered by the fact that blood is composed RBCs, which exceed the number of WBCs by a ratio of greater than 10,000:1. Separations are further hampered by the viscosity of whole blood, due to the serum and the density of cells per cubic centimeter.
A number of principles have been applied to separate RBCs from various populations of WBCs. Traditionally, gradient separations have been used. Gradient separations work on the principle that RBCs are small and dense, and can form a pellet when centrifuged, usually below a “cushion” of a substance such as Ficoll. Although effective, the gradient methods are typically slow, difficult to automate, not reproducible, give poor yields, and produce cells with poor viability. Also, the final product often contains remaining RBC contamination that requires additional processing steps (e.g., RBC lysis) to remove the inadequately separated cells.
Another commonly used method to separate blood for functional assays and clinical diagnostics is direct RBC lysis. The principle of this method is that the RBCs are more sensitive to changes in the osmolarity of the media than WBCs, such that a brief and fast change in the osmolarity of the medium or buffer will lyse more RBCs than WBCs. Indeed, lysis methods work, but as with gradient methods, the reproducibility of the separation and the viability and numbers of the remaining WBCs cells are typically poor and not representative of the whole population. Damage to WBCs can also occur with these lysis methods.
A more recently developed technology to separate subpopulations of blood cells has utilized magnetic particles to separate blood cells. This typically works by one of two principles. The first method involves positive selection by the use of magnetically labeled antibodies that bind to cell surface markers of the desired WBCs (or a cellular subpopulation of WBCs, e.g., T cells, B cells, NK cells, monocytes, dendritic cells, granulocytes, or leukocytes). The second method involves negative selection by the use of magnetically labeled antibodies to cell surface markers of RBCs or other populations to be removed. Although these methods often produce higher quality WBC preparations than gradient separations or lysis methods, it is still difficult to reproducibly obtain a WBC preparation from whole blood with high viability, yield, and purity. Specific methods for magnetic separation of WBCs are described, for example, in U.S. Pat. Nos. 4,910,148; 5,411,863; 6,417,011; and 6,576,428. Kits for performing magnetic separations are commercially available, e.g., from Dynal Biotech (Oslo, Norway) and Miltenyi Biotech (Bergisch Gladbach, Germany).
Most blood protocols utilize enrichment of WBC preparations prior to the application of magnetic particles. Such enrichment procedures can involve gradient separations, RBC lysis, or retrieval of a minor portion of the total WBC populations by buffy coat harvests from drawn blood. These partially enriched preparations of cells are then subsequently used with the magnetic particles for positive or negative selections.
Harvesting white blood cells or white blood cell subpopulations from whole blood can be challenging due to the higher abundance of red blood cells compared to that observed in bone marrow, lymph node or splenic preparations.